Further purification of RNA-dependent RNA polymerase will be undertaken. Sucrose density gradient procedures will be used to separate viral replicase activity from the large number of other host proteins. Comparison of in vitro translation products (e.g., by peptide mapping) and the purified replicase will be undertaken. If we were able to extensively purify and characterize the enzyme, we will attempt to determine binding sites of the protein to the viral RNA. Further characterization of the reaction products by hybridization to cDNA copies of virion RNA will determine if plus strand RNA is synthesized by the enzyme. Individual native and modified RNAs will be used both in the in vitro polymerase system and used to infects barley protoplasts in permitting comparison of in vitro and in vivo functions. Infected protoplasts will be used for the isolation of radioactive replicase used in proposed binding site studies. Isolated cDNA clones to BMV RNAs 1, 2, 3, and 4 will be characterized and used for hybrid-selection of entirely pure RNA components.